1. Field of the Invention
The invention relates to conjugates of tumor specific antibodies with cytotoxic compounds. The preferred conjugates contain maytansinoid compounds linked to an anti-integrin antibody by a disulfide linkage.
2. Background of the Invention
There have been numerous attempts to improve the efficacy of antineoplastic drugs by conjugating such drugs to monoclonal antibodies (Mabs) against tumor-associated antigens in order to elevate local concentration of the drug by targeted delivery to the tumor. Conversely, the potential for antibodies to actually destroy tumor cells is limited to those antibodies directed to blocking proliferative stimuli, such as the growth factors EGF and Her-2 by blocking the ligand binding to the receptors or blocking signaling to of the receptors (ErbB1 and ErbB2) or those that elicit effector functions (ADCC or CDC). Therefore, a product combining the specificity of a Mab with the killing potential of a metabolic poison has been sought. Examples of the former are doxorubicin conjugated Mab BR96 (Braslawsky, et al. Cancer Immunol Immunother 33:367-374, 1991) and pseudomonas exotoxin fused to anti-growth factor antibodies or fragments (Kreitment, et al., Intemat. J. Immunopharm. 14(3):465-72, 1992).
These attempts have encountered unforeseen limitations, such as the requirement for relatively high intracellular concentrations of the toxin compared to the number of external binding sites per cell. If the number of tumor-associated antigens on the cancer cell surface is estimated to be 105 molecules/cell, the cytotoxic agents that can be effectively used in these conjugates must have an IC50 value of 10−10-1011 M against target cancer cells. (Chari, R. V. J. Adv. Drug Delivery Rev. 1998, 31, 89-104). Secondly, the drug must either be released upon binding to the target and penetrate the cell or the entire construct must be transported into the cell and toxin cleaved or otherwise activated there.
Some of these drawbacks can be solved to a greater or lesser extent by using a highly potent drug conjugated to an internalizing antibody and using a chemical bond which has enhanced lability under intracellular conditions. Chari et al (Cancer Res. 52:127-131, 1992; Liu et al., Proc. Natl. Acad. Sci USA 93:8618-8623, 1996; U.S. Pat. No. 5,208,020) developed antibody conjugates wherein the antibody is linked to a maytansinoid via a disulfide linkage.
Maytansinoids are plant derived anti-fungal and anti-tumor agents. The isolation of three ansa macrolides from ethanolic extracts of Maytenus ovatus and Maytenus buchananii was first reported by S. M. Kupchan et al. and is the subject of U.S. Pat. No. 3,896,111 along with demonstration of their anti-leukemic effects in murine models at the microgram/kg dose range. Maytansinoids, however, have unacceptable toxicity, causing both central and peripheral neuropathies, and side effects: particularly nausea, vomiting, diarrhea, elevations of hepatic function tests and, less commonly, weakness and lethargy. Therefore, it has been a focus of research for some time to find the correct targeting moiety along with a suitable chemical process to form a maytansine-antibody conjugate with acceptable half-life of degradation.
In contrast to the high cytotoxicity of free maytansinoid, an antibody conjugate has a toxicity which is several orders of magnitude lower on antigen-negative cells compared to antigen-positive cells. The linkage by disulfide bonding has the advantage that these bonds are readily cleaved inside the target cells by intracellular glutathione, releasing highly toxic free drug. This approach has been applied to antibodies against tumor-associated antigens, for example the C242-DM1 conjugate (Liu et al., Proc. Natl. Acad. Sci USA 93:8618-8623, 1996), and HuN901-DM1 (Chari et al., 2000). However, the application of these conjugates is restricted due to the limited expression of the respective target antigens.
There is, therefore, still the need to improve this approach by using antibodies targeted to the more highly expressed tumor-associated antigens, and optionally, antigens highly expressed during the proliferative and metastatic stages of the malignancy, thus allowing for a natural concentration of toxin to the most virulent cells.
Anti-Integrin Monoclonal Antibodies
Considerable evidence shows that progressive tumor growth is dependent upon angiogenesis, the formation of new blood vessels, to provide tumors with nutrients and oxygen, to carry away waste products and to act as conduits for the metastasis of tumor cells to distant sites (Gastl et al., Oncol. 54:177-184). Recent studies have further defined the roles of integrins in the angiogenic process. During angiogenesis, a number of integrins that are expressed on the surface of activated endothelial cells regulate critical adhesive interactions with a variety of ECM proteins to regulate distinct biological events such as cell migration, proliferation and differentiation. Specifically, the closely related but distinct integrins αVβ3 and αVβ5 have been shown to mediate independent pathways in the angiogenic process. An antibody generated against αVβ3 blocked basic fibroblast growth factor (bFGF) induced angiogenesis, whereas an antibody specific to αVβ5 inhibited vascular endothelial growth factor (VEGF) induced angiogenesis (Eliceiri, et al., J. Clin. Invest. 103: 1227-1230 (1999); Friedlander et al., Science 270: 1500-1502 (1995)). Therefore, integrins and especially the alpha V subunit containing integrins, are reasonable therapeutic targets for diseases that involve angiogenesis such as disease of the eye and neoplastic disease, tissue remodeling such as restenosis, and proliferation of certain cells types particularly epithelial and squamous cell carcinomas.
Antibody Drug Conjugates
Conjugates of cell binding agents with the highly cytotoxic maytansine has been described (U.S. Pat. Nos. 5,208,020 and 5,416,064; R. V. J. Chari et al., 1992 Cancer Res. 52:127-131). Certain reagents or reactants such as N-hydroxysuccinimidyl esters (NHS) for reaction with protein amine groups have been developed for use in forming drug-protein conjugates. Reagents of this type were generally described by Carlsson et al. (Biochem J. 173: 723, 1978 and in U.S. Pat. No. 4,149,003. Nitro-pyridyl linker reagents for maytansine conjugation to Mabs and other proteins are disclosed in WO2004/016801.
In the above referenced processes, the cell binding agents are modified with a bifunctional agent such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) to introduce an active disulfide moiety. Reaction with a thiol-containing cytotoxic drug provides a conjugate in which the cell binding agent, such as a monoclonal antibody, and drug are linked via disulfide bonds. It was found that the C-3 hydroxyl position could be modified without loss of activity, in fact, certain esters were found to have enhanced cell killing activity (See Cassady, et al. Chem Pharm Bull 52(1): 1-26, 2004 for a review). U.S. Pat. Nos. 5,208,020 and 5,416,064 specifically teach the use of the activated maytansol ester of N-methyl-N-(3-methyldithiopropanoyl)-L-alanine. The maytansoid moiety from this reaction, and which is released upon reductive cleavage of the disulfide bond, has been designated DM1 [N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine, CAS Reg. No. 139504-50-0]. Thus, all the conjugates prepared using the methyldithiolated form of DM1 retain an unsubstituted methylene carbon adjacent to the disulfide bond on the drug side of the conjugate (FIG. 1).
In order to enhance the in vivo stability of this disulfide link, it is important to provide sterically hindered disulfide bonds as has been noted previously (Thorpe, et al. Cancer Research 47:5924-31, 1987). This objective can be achieved by using cross-linkers that bear one or two methyl substituents on the carbon atom adjacent to the disulfide bond or using activated drugs bearing at least one substituent on the alpha-carbon atom adjacent the sulfhydryl or disulfide substituent.
While the problems of targeted delivery are now clearly recognized, finding a suitable combination of antibody specificity and affinity, conjugation chemistry, and toxin is unpredictable. It is the object of the present invention to provide novel antibody maytansine conjugates wherein the antibody is directed to cell surface antigens sufficient in number to deliver a cytocidal dose of a maytansinoid and which conjugate has appropriate chemical and biologic stability to provide a therapeutically effective rate of release when administered to a subject.